Infectious agents, such as viruses, have evolved mechanisms and strategies to invade their hosts and to escape their immune response. Various publications have demonstrated the immunosuppressive properties of proteins encoded by viruses: the Epstein Barr human herpes virus 4 (Suzuki et al. 1995. J. Exp. Med. 182, 477-486; Qin et al. 1996 J. Immunol. 156, 2316-2323), the Mason-Pfizer monkey virus (Blaise et al. 2001 J. Gen. Virol. 82, 1597-1600), the Moloney murine leukaemia virus (Mangeney and Heidmann. 1998. Proc. Natl. Sci. USA. 95, 14920-14925) and others (see review Alcami et al. 2002 EMBO reports. 3(10), 927-932). This may be confirmed by the fact that infection by retroviruses is frequently associated with dysfunctions of the immune system of the host.
These immunosuppressive effects include the inhibition of interleukin-2-dependent lymphocyte proliferation, of the cytolytic activity of human natural killer cells, and of monocyte-medicated tumor cell killing as well as modulation of cytokine synthesis.
In vivo tests demonstrated that inactivated viruses, as well as synthetic peptides similar to retrovirus envelope proteins have immunosuppressive properties (Oostendorp et al. 1993 Crit. Rev. Oncol. Hematol. 14, 189-206; Haraguchi et al. 1997 J. Leukocyte Biol. 61, 654-666). More recently, Mangeney et al. (1998. Proc. Natl. Sci. USA. 95, 14920-14925) showed that murine tumoral cells from C57BL/6 strain, expressing a retroviral envelope protein, form tumours when injected in Balb/c mice (allograft), whereas the same cells, which do not express the retroviral envelope protein, are rejected. By carrying out different deletions in the envelope protein, a domain responsible for the immunosuppressive function that was called ISU (for “immunosuppressive”) domain, was identified.
The ISU domain was first identified in the transmembrane moiety of the envelope glycoprotein. The env (envelope) gene of retroviruses encodes a precursor polypeptide which is then cleaved into two proteins: the surface glycoprotein (SU) and the transmembrane subunit (TM). The SU protein is responsible for the recognition and the binding to the cellular receptor for the virus. The TM moiety is involved in anchoring the envelope complex (SU and TM) to the target cell membrane, and is directly responsible for cell membrane fusion and virus entry.
The structure of the TM subunit has been elucidated for many viruses, especially for the Moloney murine leukaemia virus (Mo-MuLV), the human immunodeficiency virus 1 (HIV-1) and the human T-cell leukaemia virus type 1 (HTLV-1). A highly conserved organization in the envelope proteins has also been found in non-retroviral proteins, such as those of influenza virus and Ebola virus.
Immunosuppressive effects have also been discovered in another class of proteins, characterized in the ERVs, especially HERVs (Human Endogenous Retroviruses). HERVs comprise elements which are sequences of retroviral origin that have spread into the human genome, and represent proviral remnants of ancestral infections. Therefore, strong similarities can be inferred between HERVs and retroviruses. Some of these HERV elements are still functional and can encode active proteins, i.e., viral-like proteins although most of them have accumulated mutations, deletions and/or truncations.
A role for these functional HERVs has been proposed, including a protection against retrovirus infection (Best et al. 1997 Trends Microbiol. 5, 313-318) or a protection of the foetus against the maternal immune system via immunosuppressive effects (Cianciolo et al. 1985 Science 230, 453-455; Mangeney and Heidmann 1998 Proc. Natl. Sci. USA. 95, 14920-14925). An HERV encoding an envelope protein having immunosuppressive properties was identified by Mangeney et al. (2001 J. Gen. Virology 82, 2515-2518). This publication reports that the protein encoded by HERV-H allows the envelope-expressing cells to escape immune response and to proliferate, whereas the same cells transfected with empty vectors are normally rejected by engrafted mice.
Other ERVs, especially HERVs, encoding functional envelope proteins were identified, which have fusogenic properties, i.e. are able to form syncytia in vitro (multi-nucleate cells): they include HERV-FRD and HERV-W (Blond et al. 2000 J. Virol. 74, 3321-3329; Blaise et al. 2003 Proc. Natl. Acad. Sci. 22, 13013-13018). Moreover, in vivo experiments have shown that when co-expressed with MoMLV viral particles deficient for the production of their own envelope protein, the HERV-W envelope protein can form functional viral particles, capable of infecting human cells (Patience et al. 1998 J. Virol. 72, 2671-2676). In conclusion, HERV-W has conserved its fusogenic and infectiosity properties. Analog fusogenic and infectious properties have been observed for HERV-FRD.
The observed immunosuppressive effects may be related, depending on the context, on the one hand to a virulent viral infection and on the other hand to an active proliferation of tumour cells, in mammals and particularly in human. Active proliferation of tumour cells is especially a consequence of expression of ERV viral-like proteins. However, whereas more insights are needed to completely understand the mechanisms of immunosuppression, the identification of these immunosuppressive proteins opens new perspectives for therapeutic, including vaccinal, strategies against viral infections, against induction of expression of endogenous retroviruses, or against their detrimental consequences in a host.
Vaccines currently used can especially be classified as follows:                live attenuated vaccines (bacteria or virus vaccine) consisting in an attenuated or weakened, modified pathogen. After administration to the host, the modified pathogenic organism replicates in the host and stimulates an immune response. This type of vaccine generally produces a long-lasting immunity upon single dose administration, but may cause side effects, i.e. a mild case of the illness caused by said pathogen, and thus should not be given to people with a weakened immune system.        inactivated or killed vaccines, consisting in killed or inactivated pathogen, especially as a result of heat and/or chemical treatments (whole organism). Such treated pathogens cannot replicate, and cannot cause the disease they normally raise. Therefore, they are safe and can be administered even to hosts whose immune system is weakened. However, they are not usually as effective as live vaccines and therefore require multiple dose administration.        vaccines consisting in antigenic fractions of a pathogen organism, including whole proteins or antigenic determinants thereof, especially obtained by recombinant technologies, as a result of the expression of genes encoding the antigen. The expressed protein can be administered to a patient, or the gene encoding the protein can be inserted into an expression vector which is administered to the host. Such vaccines however are usually not as effective as live vaccines and therefore require multiple doses.        
Principles applied for the design of compounds suitable for vaccine preparations capable of eliciting an immune response in a host, in order to protect a host from infection due to pathogens, including viruses, bacteria or others, have been transposed to the design of compounds suitable for treatment of established infections, by immunotherapy. Efficiency of such compounds has however not proved to be sufficient enough, especially in the field of anti-viral or anti-viral-like prophylaxis or immunotherapy. Moreover, the use of compounds still raises many issues regarding safety.
One drawback observed in the use of some retroviral envelope proteins for immunisation, either as vaccine principles or for immunotherapy, lies in their immunosuppressive properties which can prevent or affect the efficiency of the host's immune response. Consequently these proteins cannot be administered to a patient in their native form because of their potential inhibition of the immune response. A great challenge would hence be to suppress or modulate the immunosuppression properties of these proteins, without altering their antigenic properties and/or their properties related to host cell infection. However, attempts to mutate the envelope protein complex, have led to a strong alteration of its fusion and infection functions and therefore of their interest as active principle to raise an immune response (Delamarre et al. 1997 J. Virol. 71(1), 259-266; Rosenberg et al. 1999 J. Cell Biol. 145, 57-68).
This is a purpose of the present invention to identify determinants of the immunosuppressive properties of proteins, including to identify polypeptide sequences and amino acid residues involved in the modulation of the immunosuppressive properties of proteins, particularly viral or viral-like proteins, which substantially retain their antigenic properties of said immunosuppressive proteins.
It is a further object of the invention, to identify such determinants of the immunosuppressive properties of the protein, and to use the same for the design of polypeptides having modified, i.e., modulated immunosuppressive properties.
Another object of the present invention is to provide such polypeptides, which are derived from an antigenic and immunosuppressive protein, which polypeptides are characterized by modulated immunosuppressive properties while retaining antigenic properties of the starting protein.
This is also an object of the present invention, to provide means to promote an efficient immune response against pathogen organisms, especially against viruses, i.e., a cell-mediated and/or humoral immune response which would be protective against infection by such pathogen organisms, especially viruses, or protective against their detrimental effects in the host, or protective against the detrimental consequences of expression of endogenous retroviruses in a host, with reduced risks of immune system alteration. The invention also provides means suitable for treatment by immunotherapy, of patients infected with pathogen organisms including viruses, or for treatment of their detrimental effects, including malignant effects or for the treatment of patients suffering from pathologies associated with induction of the expression of endogenous viruses which are normally silent in hosts.